. Store-operated calcium entry (SOCE) constitutes a major calcium entry pathway in mammals to control lymphocyte activation, muscle contraction, gene expression and cell metabolism. The calcium release- activated calcium (CRAC) channel composed of ORAI-STIM represents a prototypical example of SOCE in lymphocytes. The clinical relevance of SOCE is exemplified by two human diseases, the severe combined immunodeficiency (SCID) and tubular aggregate myopathy (TAM), which are caused by loss- or gain-of-function mutations in ORAI1 and STIM1, respectively. Augmented SOCE is also implicated in cardiovascular disorders and cancer metastasis. Therefore, CRAC channel has been pursued as an attractive drug target for therapeutic intervention. Tremendous efforts have been directed to establish ORAI-STIM as the minimal two-component system to couple ER calcium store depletion with calcium influx across the plasma membrane. The regulatory machinery dedicated to the ORAI-STIM signaling, nonetheless, still remains incompletely defined. In this proposal, the PI aims to bridge this critical knowledge gap by unveiling the functions of two novel SOCE modulators, which reside at distinct subcellular locations to act on different steps of ORAI-STIM signaling: the initial activation of STIM within the ER lumen and the later stabilization of ORAI-STIM complexes at ER-PM membrane contact sites (MCS), where the close appositions of two membranes are separated by a gap distance of 10-30 nm. In Aim 1, based on preliminary findings from proteomic profiling of potential STIM1 interactors within the ER lumen, the PI will define how a previously-unrecognized multiple EF-hand protein cooperates with the luminal domain of STIM1 (EFSAM) to shape the activation and deactivation kinetics of SOCE. The PI will employ a new ?ER-to-PM? trafficking strategy to expose the luminal domain toward the extracellular side, thereby overcoming a major impediment to studies on the liminal sides of ER-resident signaling proteins. In Aim 2, capitalizing on the discovery of a TMEM family protein as a regulator of calcium influx at ER-PM MCS, the PI will define how this modulator responds to physiological stimuli to remodel the assembly of ER-PM junctions and PIP homeostasis to sustain SOCE. The generation of innovative optogenetic tools and a transgenic mouse model to dissect calcium signaling and protein-PIP interactions will further accelerate our structure-function relationship studies on these novel regulators. Overall, the new mechanistic insights gained through the proposed study will lead to advances in the constantly-revitalized field of calcium signaling, and in parallel, spawn the vibrant field of membrane contact sites. In the long run, discoveries made in the study can be translated into the development of effective therapeutics targeting aberrant calcium and phosphoinositide signaling.